GSTs介导马唐抗高效氟吡甲禾灵的代谢机理研究

Digitaria sanguinalis is a monocot weed in the Poaceae family, which serious impacts on the yield and quality of crops. Resistance of Digitaria sanguinalis to Haloxyfop-P-methyl was found by the field monitoring data. Currently，target resistance and metabolic resistance are recognized as the main mechanism of resistance. On the basis of our previous research, target resistance and metabolic enzymes P450 resistance were excluded. GSTs activity was significantly enhanced, and GSTs inhibitor can significantly improve the efficiency of Haloxyfop-P-methyl to resistant biotypes. So GSTs-mediated metabolic mechanism is speculated. RNA-seq and expression profiles will be analyzed to get differential GSTs gene out of resistance biotypes and susceptible biotypes. GSTs genes involving resistance are introduced into susceptible Digitaria sanguinalis by agrobacterium-mediated method. The function of GSTs genes involving resistance are verified by means of RNAi and overexpression. It will be clarified that mechanism of metabolic resistance of Digitaria sanguinalis to Haloxyfop-P-methyl mediated by GSTs, which provides a scientific basis for resistant weed management.

马唐是危害农作物最为严重的禾本科杂草之一，严重影响作物的产量和品质。田间监测发现马唐对棉田除草剂当家品种高效氟吡甲禾灵产生了抗药性。目前靶标抗性和代谢抗性被公认为抗性产生的主要原因。本项目前期研究排除了靶标抗性和代谢酶P450代谢抗性，发现抗性马唐中GSTs活力显著增强，且GSTs抑制剂能显著提高高效氟吡甲禾灵对抗性马唐的药效，推测可能为GSTs介导的代谢抗性。申请者拟以抗性、敏感生物型马唐为材料，通过RNA-Seq技术挖掘参与抗性的GSTs基因，采用农杆菌介导法将抗性基因转入敏感马唐，通过RNAi技术和超量表达技术验证抗性基因的功能，揭示GSTs介导马唐代谢抗性的机理，为制定抗药性杂草治理策略提供科学依据。

马唐（Digitaria sanguinalis）是危害旱地农作物最为严重的禾本科杂草之一，严重影响作物的产量和品质。本项目前期研究发现了棉田马唐对除草剂高效氟吡甲禾灵产生了GST介导的代谢抗药性。为探明其抗药性机理，本项目以高效氟吡甲禾灵抗性、敏感马唐为研究对象，通过对马唐转录组水平的高通量测序，挖掘到5个表达量差异显著的GSTs候选基因，其中4个基因属GSTs同工酶家族中的tau（U）族，1个基因属phi（F）族。利用RACE技术结合PCR技术克隆得到了每个基因的全长序列。为验证候选基因的功能，构建pET-32a(+)-GSTs原核表达载体并转化大肠杆菌，经IPTG诱导后发现pET-32a(+)-GST1和pET-32a(+)-GST4融合蛋白对除草剂高效氟吡甲禾灵具有体外代谢活性。构建pBWA(V)HS-GST1和pBWA(V)HS-GST4真核表达载体并遗传转化水稻日本晴，发现转基因植株对高效氟吡甲禾灵的敏感性降低，对高效氟吡甲禾灵产生了抗药性，通过HPLC检测发现高效氟吡甲禾灵在转基因水稻植株中的降解率显著高于野生型水稻，明确了GST候选抗性基因的功能，揭示了GSTs介导马唐抗高效氟吡甲禾灵的机理，为抗药性杂草治理和科学合理使用除草剂提供科学依据。